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* Department of Primary Industries, Ellinbank, Victoria, Australia
Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada
Dexcel, Private Bag, Hamilton, New Zealand
AgResearch, Grasslands Research Centre, Tennent Drive, Palmerston North, New Zealand
# Faculty of Land and Food Resources, University of Melbourne, Victoria, Australia
1 Corresponding author: chris.grainger{at}dpi.vic.gov.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: chamber • dairy cattle • methane • sulfur hexafluoride
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Studies with beef cattle and sheep indicate that CH4 estimated with the SF6 tracer technique is 93 to 95% of that measured using whole-animal chambers (Johnson et al., 1994; Ulyatt et al., 1999; McGinn et al., 2006) and 105% of that measured using hood chambers (Boadi et al., 2002). The lower estimates using the SF6 tracer technique are partly explained by the CH4 released via the rectum (Murray et al., 1976). No comparisons between SF6 and chamber techniques have been made with dairy cows at higher intakes and including rectal methane.
The objective of this study was to compare the SF6 tracer gas technique to the chamber technique for measuring total enteric CH4 emissions from lactating dairy cows. The use of the SF6 tracer gas technique within the chambers enabled a direct comparison between the 2 techniques and included CH4 both respired and released from the rectum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Animal Husbandry
Inside the chambers, cows were restrained by neck halters anchored to the floor. The harnessing device permitted feces and urine to be collected at the rear of the cow into separate collection vessels that were emptied each day. The apparatus allowed the cow to lie down on a padded mattress within the stall. Windows between the chambers enabled visual contact with the cow in the adjacent chamber and the surrounding environment.
Cows were milked twice daily using a portable milking apparatus in both the metabolism stalls and the chambers. Milking and feeding the cows in the chambers necessitated opening the chamber doors, thereby disrupting gas measurements. Milk weights were measured and subsampled into bronopol (0.5% wt/wt) preservative and analyzed for concentrations of fat and protein using a near-infrared milk analyzer (model 2000, Bentley Instruments, Chaska, MN).
The bulk of the cows’ diet was fresh ryegrass pasture that was harvested daily. When cows were in the metabolism stalls or chambers, fresh pasture was placed in the feed bins twice daily to ensure ad libitum intake. When cows were in the chambers, the feed was provided while the chamber doors were open for milking. Cows also received 5 kg/d of cracked barley (as-fed basis) in 2 feedings. All feed offered and refused was weighed daily. Samples of feed and refusals were dried to determine DM content, and total daily DMI was calculated per cow (Table 1
). Representative samples of the pasture and grain were collected daily and pooled to form 4 samples of each feed over the 32-d measurement period. The samples were oven dried and ground through a 0.5-mm sieve, then analyzed by near-infrared spectroscopy by a commercial laboratory (FeedTest, Hamilton, Victoria, Australia). The pasture contained 71.1 ± 1.2% apparently digestible DM, 16.4 ± 1.0% CP, and 54.4 ± 0.9% NDF, on a DM basis. The grain contained 85.8 ± 2.2% apparently digestible DM, 10.7 ± 0.3% CP, and 6.6 ± 0.8% ADF.
Chamber Design and Operation
Each chamber, located in an open barn, had a volume of 41.5 m3, with inner surfaces of stainless steel, except for a concrete floor (Figure 1). The air within each chamber circulated independently of the other chamber at about 7.2 m3/min. The circulated air was filtered and maintained at a set relative humidity (55% in this experiment), and temperature (20°C in this experiment). The condensed water from the dehumidifier was collected in an open container inside the chamber, allowing dissolved gases to vent back into the chamber. Air was recirculated and reentered the chamber at the front of the animal stall, with an exhaust rate of approximately 1 m3/min, to the outside of the barn. The exhausted air stream first passed through straight polyvinyl chloride (PVC) duct (50 mm i.d.) to generate a laminar flow. Flow rate was measured using a turbine meter (SP2-CB-H7-A 4X, Sponsler Inc., Westminster, SC) and the data were recorded (DT800 data logger, Datataker Pty. Ltd., Rowville, Victoria, Australia) at 10-s intervals. The exhausted air was replaced with fresh air drawn in through a large-diameter PVC duct 40 m from the barn.
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). The air samples were drawn using 3 dedicated diaphragm vacuum pumps with 16 L/min flow capacity (107CD18-198, Rietschle Thomas, Seven Hills, NSW, Australia). The sample streams were continuously dried in a dehumidifier (model LGD03-4, Tation, Frigematic Industries, Melbourne, Victoria, Australia) where the dew point was set to 2°C. The condensate was removed by providing a "controlled leak" with fine capillary tubing leading from the water traps. Throughout the experiment, an automated calibration of the gas analyzer was conducted every 4 h by directing ultrapure N2 (zero) and a standard gas mixture (span) through the analyzer. The span gas had similar gas concentrations to that expected in the exhaust samples from the chambers. Each gas passed through the gas analyzer twice (4 min x 2) and the concentration recorded at 10-s intervals. The timing of the zero and span gas checks and all other analyses were controlled by the data logger. The difference between the analyzed zero and span gas concentrations and the actual zero and span concentrations were used to calibrate the gas analyzer. A primary standard gas (supplied by CSIRO Atmospheric Research Division, Aspendale, Victoria, Australia) was used before the experiment to confirm the concentration of the standard gas mixture. Any change (drift) in the gas analyzer was corrected daily, but the correction was always <1% over the duration of the experiment.
The air streams were connected to a gas analyzer (Xentra 4100C1, Servomex, East Sussex, UK) containing an infrared CH4 sensor with a range of 0 to 1,000 ppm. Sequencing of the sample streams to the analyzer was controlled by solenoids to ensure a quick response time after switching the air stream. The CH4 concentration, exhaust airflow rates, and relative humidity and temperature of the exhaust air were recorded at 10-s intervals over repeated 4-min periods. In each 12-min period, the intake air (common to both chambers) was sampled for 4 min, followed by the exhaust from chamber 1 for 4 min and then the exhaust from chamber 2 for the final 4 min. This routine was repeated every 12 min.
Chamber Emission Calculations
The measurement day for each chamber was from the time the door was closed in the morning, following milking, feeding, and the exchange of canisters used in the SF6 tracer technique, to the following morning when the door of each chamber was opened. In the afternoon, the doors were opened briefly for the second milking and feeding. For each measurement day, 3 modes were identified to facilitate chamber emission calculations. Mode 1 was the duration following door closure when the chamber air was transient; that is, CH4 concentration was increasing inside the chamber. Mode 2 was the duration of the measurement day that the chamber air was in a steady-state condition, and mode 3 was designated as the duration the doors were opened (afternoon). In mode 3, the CH4 emissions were assumed to be equal to the previous CH4 emissions just before the door was opened. For the remaining modes 1 and 2 when the doors were closed, the CH4 emission (F; g) was determined from a storage component and a flow-through component. The storage component accounted for the transient condition of the air in the chamber (mostly during mode 1 just after the door was closed), while the flow-through component accounted for the loss of CH4 from the chamber. The storage component (first bracketed term in equation 1) was determined from the volume of the chamber (V; m3), the difference between the CH4 chamber concentrations at time t (Ct; g/m3) and the previous measurement (Ct–1; g/m3), and the duration between sequential measurements (D). The flow-through component (second bracketed term in equation 1) was determined from the difference between the exhaust and inlet CH4 concentrations (
C; g/m3), the exhaust airflow (v; m3/min), and the duration between sequential measurements (D). The individual emissions from each sequential measurement (during mode 1, 2, or 3) were then summed over each measurement day (from door closing to door opening next morning) to give the total emission during each measurement day:
 | [1] |
The accumulated CH4 emission (
F; g) over each measurement day was corrected using calibration data collected automatically every 4 h. While the calibration of the gas analyzer was being conducted, the chamber gas measurements were interrupted. However, the emission during each calibration was accounted for by adding the calibration duration to the first calculation of emissions following the calibration.
The CH4 concentration (C; g/m3) was calculated from the mixing ratio (Ratio; µmol/mol) given by the gas analyzer, using the ideal gas law:
 | [2] |
where MW is the molecular weight of CH4 (16), 1,000 is the conversion for L/m3, P is the air pressure (atm), R is the gas constant (0.08205 L atm mol–1 K–1), and T is the temperature (K). In this calculation, only the mean of the last 12 of the available 24 data values from each 4 min cycle was used, which ensured the previously sampled air was purged from the system.
Calibration of the Chambers
The accuracy of the chambers was checked before starting each period by comparing a known release of ultrapure (>99.9%) CH4 (Linde Gases Pty. Ltd., Thomastown, Victoria, Australia) to that calculated using equation 1, once steady-state conditions were reached. The rate of gas release was controlled using a mass flow controller (MFC series 100, Sierra Instruments Inc., Monterey, CA).
The chamber calibrations involved 3 stages. In stage 1, the chambers were operated with no source of gas (empty chamber and no gas release). This allowed evaluation of any bias error in the analytical system. Stage 1 tested that the magnitude of the calculated emission was indeed zero when there was no emission inside the chamber, and therefore that there was no significant offset (bias) in measurement system.
In stage 2, the mass flow controllers were used to release CH4 into the exhaust duct at location 3 in Figure 1. The inlet sample line was moved from location 1 to location 2 and the sample line normally at 2 was moved to location 4. In this manner, we could perform a near-instantaneous evaluation of the entire analytical system that included the turbines, sample lines, connections, solenoid valves, pumps, gas analyzer, data recorder, and the calculation of the flux rates. It was expected that near-perfect recovery should result from this test, because leakage of gas along the exhaust duct was unlikely.
In stage 3, all sampling lines were reverted back to their normal positions. The release of CH4 was switched from location 3 to location 5 (inside the chamber). This configuration allowed the whole chamber to be included in the recovery test. With stage 3, the time required to reach a new equilibrium concentration with a constant injection rate from the mass flow controllers was 90 min. A total of 8 calibrations using stages 1, 2, and 3 were made for both chambers to characterize the recovery rates.
SF6 Tracer Technique for Respiratory CH4 Measurement
The SF6 tracer technique has been used extensively to measure CH4 production from sheep and cows in grazing and confined situations in New Zealand (Woodward et al., 2006) and elsewhere. The technique requires air sampled around the nostrils to be accumulated in an evacuated PVC canister placed around the cow’s neck (Johnson et al., 1994). Air is drawn continuously over a 24-h period into the canister through a tube positioned near the nostrils of the cow. The continuous airflow rate is controlled by passage through a capillary tube so that approximately 0.8 mL/min is accumulated over 24 h, after which the canister is replaced.
A permeation tube containing SF6 was placed into the rumen of each cow about 3 mo before the measurements reported here. The permeation tubes were manufactured in December 2004 by the National Institute of Water and Atmospheric Research, New Zealand, and were filled with about 2.3 g of SF6. The release rate of SF6 was predetermined over a 10-wk period by weighing each permeation tube weekly; the average release of SF6 was 3.7 ± 0.7 mg/d.
The concentration of CH4 and SF6 in the canisters was analyzed by gas chromatography (Shimadzu 2010, Shimadzu Corp., Kyoto, Japan and Hewlett-Packard 5890, Hewlett-Packard Labs, Palo Alto, CA), fitted with an electron capture detector (350°C) to determine SF6, and a flame-ionization detector (250°C) to determine CH4 concentration. All samples were analyzed in duplicate except standards, which were analyzed in triplicate. The gas chromatograph was fitted with a 3.3-m molecular sieve column with an i.d. of 0.32 mm and film thickness of 300 µm (Alltech Associates, Auckland, New Zealand). The column and injector temperatures were both 85°C but baked out at 200°C daily. Nitrogen was used as the carrier gas at a flow rate of 40 mL/min.
Three standards prepared by the National Institute of Water and Atmosphere (Wellington, New Zealand) were used to calibrate both gas chromatographs. Standards were mixtures of SF6 and CH4 in low, medium, and high concentrations (range: 15 to 1,000 ppt for SF6; 2 to 200 ppm for CH4). The standards were run at the beginning and end of each day with the medium standard run every 10 samples throughout the day. Gas concentrations (SF6 and CH4) were determined from peak areas and identified from their different retention times relative to the known standards.
Calculation of CH4 emissions requires measurement of background SF6 and CH4 concentrations to represent inspired air, usually upwind from grazing cows. In this experiment there was no difference in either CH4 or SF6 concentration when measured from a canister hung inside the chamber to measure chamber air and a canister positioned on the cow’s neck while in the chamber. The lack of difference was due to the low exchange of air in the chamber (1 m3/min) so there was a build-up of gas in the chamber, and also because of the circulation within the chamber (7.2 m3/min). Because the CH4 and SF6 concentrations in the chambers and the canisters were similar, the chamber inlet concentration (location 1 in Figure 1) was used as a background concentration for calculating emissions from SF6 in equation 3. In this manner, the SF6 tracer technique effectively measured the same emissions as the chamber, including those respired, eructated, and released through the rectum. This was unlike the situation for chambers with a high-volume exchange of air (e.g., McGinn et al., 2006), which resulted in much lower concentrations of CH4 and SF6 in the chamber compared with the canister on the cow. In this latter case, the chamber concentrations are used as background values and the SF6 tracer technique does not measure the flux from the rectum.
The CH4 emission (QCH4; g/d) was calculated using the SF6 and CH4 mixing ratio (µmol/mol) sampled by the canisters (CSF6 and CCH4, respectively) on the cows and inlet air streams (CbSF6 and Cb CH4, respectively), and the predetermined SF6 release rate (QSF6; g/d) from the permeation tube (equation [3]) where MW is the molecular weight of the gases:
 | [3] |
Statistical Analyses
The ratio of the total daily CH4 emission measured using the SF6 technique to that measured using the chamber was calculated for each cow as a direct measure of agreement between the 2 methods. These ratio data were analyzed using a linear mixed model that included fixed effects for chamber and treatment (monensin vs. control), and linear covariates for DMI, permeation tube SF6 release rate, and actual CH4 emissions (g/d). Random effects included in the model were day (corresponding to the 3 d of measurements for each pair of cows), cow, and cow by day. Fixed effects were tested for significance in the model using sequential Wald tests. Random effects (components of variance) were tested using the 
2 change in scaled deviance for nested models. Nonsignificant fixed terms were then excluded from the model, as were nonpositive components of variance. Predicted mean ratios and corresponding SE were obtained using the simplified model. Histograms of residuals and graphs of residuals vs. fitted values were examined for nonnormality of distribution (skewness and outliers) and constant variance. Apparent outliers were further evaluated using robust Z-scores:
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where ri is the ith random effect (residual), and qp is the pth quantile of the distribution of random effects (Australian National Quality Assurance Program, 2006). The quantiles were estimated by a kernel density estimate (Silverman, 1986). This was done both for the residuals in the lowest stratum (repeated observations) and for the estimated random effects of cow. Units with large Z-scores (in this case >3.2) were removed from the analysis. All analysis was performed using GenStat (2006) software.
A mixed model was used to examine the components of variation for actual CH4 emissions (g/d) estimated using both techniques. The mixed model included fixed effects for chamber, treatment (monensin vs. control), and a linear covariate for DMI. Random effects included in the model were cow and day within cow. The estimated components of variation were used to calculate, excluding data from outliers, the CV within cow and between cows.
A meta-analysis was developed to compare the ratios observed in the present study with the ratios obtained in studies conducted in Canada (McGinn et al., 2006) and Ireland (F. O’Mara, University College Dublin, Ireland; personal communication). In both these studies CH4 emissions were estimated by chambers and by use of the SF6 tracer technique within the chambers. The meta-analysis took the form of an elaborate mixed model for the combined ratio data. The model was defined by first developing an appropriate mixed model for each data set separately, and then incorporating these into a single model. Any nonpositive variance components identified in the initial separate analyses were not included in the extended model. The simplified model described above was used for the Australian data. The Canadian data were specified initially with factorial fixed effects for feed type by intake level, and random effects for period by chamber, and repeated measures nested within chamber by period. The random effects for period and chamber were excluded from the combined model. For the Irish data, the relationship between the ratio and intake level (fasted vs. fed), was first checked graphically. The combined mixed model included only a fixed effect for the mean, and a random effect for units. The combining of the 3 simplified models into a single analysis was achieved in GenStat by specifying missing values in each factor definition when the factor did not apply, adding the respective fixed and random effects models, and specifying an option in the GenStat code to retain all units in the analysis, whether factor levels were missing.
The relationship between DMI and actual CH4 emissions (g/d) was examined for the combined Australian and Canadian data sets (those studies in which DMI was measured) by fitting a similar combined model for the ratio of CH4 (g/d) to DMI (kg/d).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Methane Emissions
The tracer technique failed on 6 out of 48 cow-days (88% successful measurements) due to breakages and blockages of air lines. Due to these missing values, mean CH4 emissions for the experiment were calculated using only the paired data (i.e., when both techniques functioned).
The accuracy of the SF6 tracer technique was investigated by considering the ratio of the CH4 emission measured using the SF6 tracer to the emission measured using the chamber for each cow on each day. In this analysis, data outliers were identified (Z-values >3.2) and removed from subsequent analysis of the ratios (Figure 2). Data outliers were found for 1 cow (over 3 d) having abnormally low chamber emissions and for a second cow (over 3 d) in which SF6 tracer emissions were abnormally high. There were no obvious detectable physiological reasons for these abnormal emissions. For the chambers, CH4 emissions averaged (mean ± SD) 322 ± 57.5 g/d (n = 36). For the SF6 tracer technique, CH4 emissions averaged (mean ± SD) 331 ± 74.6 g/d (n = 36).
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). There was also no effect of permeation tube release rate (P = 0.90) or actual CH4 emission (g/d) measured in chambers (P = 0.77) on the ratio.
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The ratio was not affected by level of CH4 emission, but was affected by level of DMI. This seems contradictory because one would expect DMI to be correlated with level of CH4 emission. However, estimates of CH4 emissions varied with DMI depending upon the technique. The linear regression of CH4 emission with DMI was 12.9 DMI (kg/d) + 72.3 (R2 = 0.39) for the chamber technique and 18.5 DMI (kg/d) – 9.5 (R2 = 0.56) for the SF6 tracer technique. On only 8 occasions (out of a total of 36 data observations) was DMI of cows above 20 kg/ d; hence, more data would be required for cows with high intakes to establish greater confidence in the relationship reported above.
The ratio of 102.3% for the 2 techniques is different from that reported previously for beef cattle and sheep, in which the SF6 technique was 5 to 7% lower than the chamber (Johnson et al., 1994; Ulyatt et al., 1999; McGinn et al., 2006). Differences between techniques in those studies were partially attributed to the release of CH4 from the rectum, which was not measured by the SF6 tracer technique in those studies, but was measured by the chambers. About 1% of the total enteric CH4 emissions is released from the rectum in sheep (Murray et al., 1976), although no estimates are available for cattle. We expected that the 2 techniques in our study would give similar estimates of CH4 emissions because our in-chamber use of the SF6 tracer technique measured total enteric CH4, including CH4 both respired and released from the rectum.
Previous studies comparing the 2 techniques did not use animals emitting high quantities of CH4 (Johnson et al., 1994; Ulyatt et al., 1999; McGinn et al., 2006). The results of our study support the hypothesis that the SF6 tracer technique is accurate when used with dairy cows that have high DMI and high CH4 emissions relative to beef cattle. A qualification to this statement is that at DMI above 20 kg/d, the SF6 tracer technique may overestimate CH4 emissions.
Variability in Estimates of Methane Emissions
One advantage of the chamber technique is that it provides information on the variability of emissions within a day. For an individual cow, the emission was typically highest after feeding, and peaking at about twice the lowest values found just before feeding, as shown in Figure 3.
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The higher variability between days using the SF6 tracer technique indicates the need to replicate measurements over a greater number of days compared with chamber measurements to obtain the same level of precision as the chamber technique. This requirement would be even greater for studies conducted under grazing conditions where weather conditions are variable (Ulyatt et al., 1999). The variability among cows was substantially higher than within cows, and also showed a difference between techniques; CV of 19.6 and 17.8% for the SF6 tracer and chamber techniques, respectively. These imply a CV of 8.2% measurement contribution from the SF6 technique, excluding biological variation in actual CH4.
Using chambers, Blaxter and Clapperton (1965) reported a 7.2% CV for day-to-day variation based on 989 24-h determinations of CH4 for sheep and cattle. They also reported a CV between animals of 5.0 to 7.5% for sheep given a fixed amount of feed. The CV between animals, however, appears to be larger in chamber studies when intake is not restricted. For example, McCourt et al. (2005) reported a CV for 135 beef steers of 17.2% (DMI: 4.4 to 11 kg/d), Bruinenberg et al. (2002) reported a CV of 16.9% for 96 measurements from grass-fed dairy cows (DMI: 6.7 to 20.8 kg/d), and Yan et al. (1997) reported a CV of 18.2% for 221 dairy cows fed grass silage based-diets (DMI: 7.5 to 24.5 kg/d). Thus, the CV we report (approximately 18%) for animal variation was similar to that reported previously for cattle fed ad libitum.
Our study shows that when the SF6 tracer technique is used on an animal within a chamber, the variability among animals is larger than for direct chamber measurements after accounting for effects of DMI. Boadi et al. (2002) also reported for a small number of animals that animal variability was greater for the SF6 tracer technique than for chambers, even though the animals had similar intakes. Vlaming et al. (2005) summarized a number of studies that used the SF6 tracer technique with grazing dairy cows and sheep, and reported CV for animal variation of 31.1 and 35.8%, respectively. However, DMI was variable in that study and was not accounted for in the CV calculations. We did adjust for intake in our analysis. Our CV estimates increase to 19.2% if we do not adjust for DMI.
Meta-Analysis
Combining the data from the present experiment (Australian) with the Canadian and Irish data resulted in a data set characterized by a wide range of CH4 emissions due to the range in DMI, diet quality, and animal type (Figure 4). In the Canadian study, the chambers measured emissions from the whole animal, whereas the SF6 tracer technique only measured respired CH4. The Canadian data set (n = 46) was for growing beef cattle fed diets containing 30 to 70% concentrate (DM basis) fed at 65 or 100% of ad libitum intake. One outlier with a ratio of 153% was excluded from subsequent analysis. In the Irish study, both the chambers and the SF6 tracer technique measured emissions from the whole animal. The Irish data set was for nonlactating Holstein-Friesian cows fed a diet of 67% concentrate and 33% straw (DM basis, ranging from maintenance level of energy intake or fasted over a period of 6 d).
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). As expected, the ratio was highest for the Australian data set because both techniques measured whole-animal CH4 emissions, whereas that was not the case in the Canadian study. The lower ratio obtained for the Irish data set may be due to the very low emissions of some cows caused by fasting. With very low emissions, a difference of only a few grams per day could cause a large change in the ratio. The meta-analysis demonstrated that over a range of conditions (Australian, Canadian, and Irish studies), the SF6 tracer technique gave CH4 emission values that were about 8% lower than complete recovery of whole-animal emissions. However, this difference is also explainable in terms of the different DMI and the relationship between the ratio of SF6 to chamber CH4 and DMI, observed both within and between studies.
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). There was a difference (P < 0.001) between the slope of the Australian and Canadian data sets (17.1 vs. 20.8; SED = 0.93) when CH4 (g/d) was plotted against DMI (kg/d) with the intercept = 0. A difference between the 2 locations was expected based on the difference in diet composition for these 2 studies. Our value of 17.1 g of CH4/kg of DMI for dairy cows was slightly lower than the mean values of 18.4 to 19.8 g of CH4/kg of DMI reported by Bruinenberg et al. (2002) for dairy cows fed mostly grass in chambers, possibly due to the higher proportion of concentrate (26 vs. 10% of DMI) fed in our study.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication October 22, 2006. Accepted for publication February 22, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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